Prokaryotic Expression And Characterization Of Bluetongue Virus Vp3、5、7Protein And Peste Des Petits Ruminants Virus H Protein

Bluetongue virus ([BTV]; genus Orbivirus, family Reoviridae) is the etiologic agent of bluetongue (BT), an important and re-emerging arboviral disease of ruminants. At present, bluetongue disease is listed in A infectious disease by World Organization for Animal Health (OIE). It is seriously endangering the livestock industry in china and the development of international trade in animals and animal products. Bluetongue virus-encoded protein, VP3protein is the main core of the virion proteins, contain group-specific epitope. VP5constitute virus coat protein, to specific antigens, VP5is the unique glycosylation of proteins, glycosylation and pathogenicity of the virus concerned. VP7is one of the major structural protein in the BTV Nuclear capsid, and it contains group-specific antigenic determinants, can encourage the body to produce strong group-specific response. Therefore, Bluetongue VP7and VP3, VP5protein has become hot topics. In this study,the antigen VP3,5,7of BTV were expressed in E.coli by the recombinant vectors, and they can serve as diagnostic reagent antigen. This study gives us a clue to reaserch the diagnostic reagent and can be helpful to the development of new vaccines VLPs.Peste des Petits Ruminants is an acute or subacute, highly contagious diease of domestic and wild small rumianats, especially sheep and goats. In our country, it is classified as A class of animal disease, and it is on the FAO/OIE noticed list. In recent years, the breakout of PPR in the surrounding countries and regions of our country and Tibet Region poses a serious threat on the sheep industry. The genomic of PPRV mainly encode6the structure proteins and2non-structural proteins, H gene encoding proteins as viral and host cell membrane receptors in the binding process, adjust the virus adsorption and invaded host cells, is the main virus antigen component, is also a main protein causing host cell lesions. So study of H protein is extremely important. This study by constructing recombinant plasmid pET-32a-H, succeeded in realizing the H protein expression, for the development of PPRV vaccine and standard testing antigen laid the foundation for research. 1Expression and antigen tic characterization of the major protein VP3、5、7of Bluetongue virusAccording to published VP3,5,7gene sequences of BTV-12on GenBank, primers were designed and synthesized. The VP3,5,7gene of BTV-12were amplified by RT-PCR. Then VP3(VP3-1,VP3-2) gene of BTV were cloned into prokaryotic expression vector pET-30a and sequenced; VPS and VP7were cloned into prokaryotic expression vetor pMAL-c2x and sequenced. The recombinant plasmid pET-30a-VP3-1, pET-30a-VP3-2, pMAL-VP5and pMAL-VP7were constructed. The recombinant expression vectors were transformed into E.coli BL21(DE3) and E.coli TB1and induced by IPTG, respectively. The His-tagged recombinant protein of VP3-1and VP3-2were purified using a His-tag affinity chromatography column on Ni2+-nitrilotriacetate (NTA) resin. The fusion protein (MBP-VP5and MBP-VP7) were purified according to the instruction manual of pMAL protein fusion and purification system. SDS-PAGE and Western blotting revealed that they were all expressed with the expected molecular mass. The indirect enzyme-linked immunosorbent assay (ELISA) indicated that the expressed BTV VP5was good immunogenicity.2Expression and antigentic characterization of the major protein H of PPRV According to published H gene sequence of PPRV Nigeria/75/1on GenBank, a pair of primers were designed and synthesized. The H gene of PPRV was amplified by RT-PCR. Then H gene of BTV was cloned into prokaryotic expression vector pET-32a and sequenced. The recombinant plasmid pET-32a-H was obtained. The recombinant expression vectors were transformed into is.coli BL21(DE3) and induced by IPTG. The His-tagged recombinant H protein was purified using a His-tag affinity chromatography column on Ni2+-nitrilotriacetate (NTA) resin. SDS-PAGE and Western blotting revealed that the expressed protein was fusion protein, with a molecular mass of87ku. The expressed H protein can produce a specific reaction to sera positive for PPRV, shows highly immunogenic.